Clinical and laboratory evaluation
The severity of all participants’ clinical course was determined on each
hospital day using a modified Wang bronchiolitis severity score,
configured by the replacement of patients’ general condition with oxygen
saturation13. Oxygen saturation was considered as a
more reliable measurement than the patients’ general condition, which
was recorded by different physicians at the time of admission. Moreover,
data support the necessity of oxygen saturation measurement for
evaluating the severity of bronchiolitis14, while
general condition may be the component of Wang score which presents the
highest correlation with oxygen saturation15. Modified
Wang score ranges from 0-12 with 12 being the most severe. Scoring was
obtained as follows: respiratory rate (0:<30/min, 1:30-45/min,
2:46-60/min, 3:>60/min), use of accessory muscles (0:none,
1:intercostal recession, 2:trachea-sternal recession, 3:severe
recessions with nasal flaring), room air oxygen saturation
(0:> 95%, 1:90-94%, 2:<90%,
3:<85%), and the presence and extent of wheezing (0:none,
1:terminal expiration with stethoscope, 2:entire expiration and
inspiration with stethoscope, 3:inspiration and expiration without
stethoscope). The length of hospitalization, O2supplementation, intravenous (IV) fluid requirement, nasogastric (NG)
feeding, use of any medication and Intensive Care Unit (ICU) admission
were noted.
Serum 25(OH)D levels, as well as cathelicidin and β-defensin-2 levels in
the respiratory secretions, were measured in the Research Laboratory of
the 2nd Department of Pediatrics, National and
Kapodistrian University of Athens.
Βlood samples were centrifuged within one hour of blood sampling. Serum
samples were frozen at -20°C until further analysis. Quantitative
measurement of serum 25(OH)D levels was carried out using the following
ELISA laboratory kit: 25(OH) -Vitamin D direct ELISA Kit
(Immunodiagnostik AG). Competitive ELISA technique with selected
monoclonal antibody recognizing 25(OH)D was used.
Μeasurements of AMPs and total protein were performed at the same time
for each nasal secretion sample in order to normalize the AMPs values.
Nasal secretions were collected by vacuum-aided suction, without
chemical stimulation, in order to avoid introducing foreign substances
into the nasal fluids. Gentle manipulation of a narrow rubber-tipped
vacuum device inside the nasal passageways mildly stimulated nasal
secretions. Nasal samples were handled with Aprotinin (0.6TIU per ml of
sample) immediately after collection in order to thwart the action of
proteolytic enzymes. Secretions were stored at −20°C.
Quantitative analysis of AMPs in nasal secretions were carried out using
the following ELISA Test Kits: Human Beta Defensin-2
(EK-072-37, Phoenix Pharmaceutical) and Human LL-37 (HK321-01,
Hycult Biotechnology) according to manufacturer’s instructions. The
specificity of the kit to both human β-defensin-2 and LL-37 is 100%.
All ELISA measurements were carried out in duplicate and included
internal standards used to construct standard curves for analyte
concentration assessment.